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Phytomedicine 18 (2011) 1220– 1228

Contents lists available at SciVerse ScienceDirect

Phytomedicine

jou rn al hom epage: www.elsev ier .de /phymed

ooibos (Aspalathus linearis) offers cardiac protection againstschaemia/reperfusion in the isolated perfused rat heart

.G. Pantsi a, J.L. Marnewickb, A.J. Esterhuysea, F. Rautenbachb, J. van Rooyena,∗

Department of Biomedical Sciences, Faculty of Health and Wellness Sciences, Cape Peninsula University of Technology, Bellville, South AfricaOxidative Stress Research Centre, Faculty of Health and Wellness Sciences, Cape Peninsula University of Technology, Bellville, South Africa

r t i c l e i n f o

eywords:ooibosspalathus linearisreen tealavonoidsxidative stress

schemia/reperfusionpoptosis

a b s t r a c t

Rooibos, a unique South African herbal tea, is known to be an important source of unique polyphe-nolic compounds. In the present study we have quantified the main polyphenolic compounds in bothfermented/traditional and unfermented/“green” rooibos (Aspalathus linearis) and evaluated its cardio-protective effects against ischaemia/reperfusion injury. Male Wistar rats consumed aqueous rooibos andgreen tea (Camellia sinensis) extracts (2%, w/v) for 7 weeks before their hearts were rapidly excised andperfused in a working heart perfusion apparatus. The results showed that the rooibos supplementedhearts significantly improved aortic output recovery after reperfusion when compared to the green teasupplemented hearts. Additionally, we showed that the rooibos extracts, containing the highest amountof flavonols, significantly decreased the level of cleaved caspase-3 and PARP, both pro-apoptotic proteins,during reperfusion when compared to green tea. Green tea supplementation increased phosphorylation

of total PKB/Akt, Akt (threonine 308) and Akt (serine 473). The rooibos extracts did not cause significantchange in the levels of the pro-survival PKB/Akt (threonine 308 and serinet 473). The GSH/GSSG ratioin the hearts of the green tea supplemented group was significantly (p < 0.05) lower when compared toRF (37.78 ± 28.63), RU (33.20 ± 4.13) and C (45.50 ± 14.96). The results clearly demonstrate the cardio-protective properties of aqueous rooibos extracts via the inhibition of apoptosis which can possibly berelated to the flavonol content of this unique South African herbal tea.

ntroduction

Myocardial ischemia/reperfusion (I/R) occurs following partialr complete cessation of blood circulation to the myocardium. Thenvolvement of oxidative stress in the pathogenesis of myocardial/R injury has been previously reported, with the generation ofeactive oxygen species being one of the major mechanisms under-ying myocardial reperfusion injury (McCord et al. 1985; Klonert al. 1989; Dhalla et al. 2000). Observational studies have repeat-dly shown that diets high in plant-based foods and beveragesre associated with a lower risk of chronic diseases such as car-iovascular disease (Hertog et al. 1993; Hollman et al. 1999; Hu003). In addition, it has been reported that antioxidant-rich oilsave cardioprotective effects against IR injury (Esterhuyse et al.005; Bester et al. 2006). Flavonoids possess properties that alle-iate ischemia/reperfusion injury by helping to re-establish blood

ow in post-ischemic hearts. A number of flavonoids and polyphe-olic compounds have shown the capacity to dilate vessels (Achikend Kwan 2003; Engler and Engler 2006; Sanchez et al. 2006;

∗ Corresponding author. Tel.: +27 21 9596523; fax: +27 21 9596874.E-mail address: vanrooyenj@cput.ac.za (J. van Rooyen).

944-7113/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.phymed.2011.09.069

© 2011 Elsevier GmbH. All rights reserved.

Nishioka et al. 2007; Jochmann et al. 2008). Research has shownthat flavonols possess antioxidant, anti-inflammatory and vasore-laxant activities (Chan et al. 2000; Perez-Vizcaino et al. 2006).Epidemiological studies report an inverse association betweendietary flavonol intake and mortality from coronary heart disease(Geleijnse et al. 2002; Lin et al. 2007).

According to the World Health Organization, approximately 80%of the world’s population currently relies on indigenous or tra-ditional medicines for their primary health needs. Most of thesetherapies involve the use of aqueous solutions of plant extracts(Zhang 2002). Rooibos is a herbal tea made from the leaves andstems of the indigenous South African plant, Aspaluthus linearis(Brum.f) Dahlg. (family Fabaceae; tribe Crotalarieae) (McKay andBloemberg 2007; Marnewick 2009). The use of herbal preparations,typically prepared by steeping or heating the crude plant mate-rial, has prevailed for centuries and healthcare providers in SouthAfrica and worldwide today often recommend herbal preparations.Rooibos is gaining popularity as a health/functional beverage bothlocally and worldwide (Joubert et al. 2008; Joubert and Schulz

2006). This is mainly due to the natural absence of caffeine andlow tannin content (Blommaert and Steenkamp 1978; Galasko et al.1989). Rooibos is a good dietary source of antioxidants containingmostly flavonoids such as flavonols, but also the unique C–C linked

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ihydrochalcone glucoside, aspalathin (Koeppen and Roux 1965)s well as cyclic dihydrochalcone, aspalalinin (Shimamura et al.006). Studies have reported on the in vivo and in vitro antiox-

dant (Yoshikawa et al. 1990; Gadow et al. 1997a,b; Marnewickt al. 2003; Joubert et al. 2004), anti-inflammatory properties (Babat al. 2009) as well as the modulation of oxidative stress by rooi-os (Ulicna et al. 2006; Fukasawa et al. 2009; Marnewick et al.011). Beltrán-Debón et al. (2011) found rooibos beneficial effects

n a hyperlipidemic mouse model when they fed 10 g/l of rooibosxtract for 14 weeks. They speculated that the possible mechanismay be activation of AMPK. These results motivated to consider

he possible use of rooibos as a natural therapeutic substanceo manage metabolic disease. No experimental data have beenublished to demonstrate the chronic intake effect of rooibos on

schaemia/reperfusion injury in an ex vivo rat heart model. Basedn results from previous in vitro, animal and human studies, theim of the present study was to determine whether dietary rooi-os supplementation could protect against ischaemia/reperfusion

njury in the isolated perfused rat heart. The possible biochemicalechanisms of protection are also addressed.

aterials and methods

hemicals

Rutin, quercetin and luteolin were purchased fromigma–Aldrich (Johannesburg, South Africa). Orientin, isoori-ntin, vitexin, isovitexin, hyperoside and chrysoeriol wereurchased from Extrasynthese (Genay, France). Aspalathin was aift from the South African Medical Research Counsel (PROMEC,rof WCA Gelderblom). Methanol, dimethylsulfoxide (DMSO) andrifluoroacetic acid were purchased from Merck (Johannesburg,outh Africa). Standards were dissolved in methanol and DMSO1 mg/ml) as per the instructions of the manufacturer and storedt −40 ◦C. Aliquots of 20 �g/ml were injected into the HPLC.

Phenylmethanesulfonyl fluoride (PMSF), sodium fluoride (NaF)nd sodium orthovanidate (Na3VO4) were all obtained fromigma–Aldrich, SA. Sodium dodecyl sulfate polyacrylamide gellectrophoresis (SDS-PAGE), polyvinylidene fluoride (PVDF) mem-ranes and tris-buffered saline with 2% tween (TBST) were allbtained from Biorad. Antibodies from Cell Signaling were also usednd protein bands on the gels were quantified using the UnscanKitrogram.

ooibos and green tea preparation

Aqueous extracts were prepared by the addition of freshly boiledap water to the tea leaves and stems of unfermented/“green” andermented/traditional rooibos (Superior grade, generous gift from

r. Arend Redelinghuys, Rooibos Ltd., Clanwilliam, South Africa)nd green tea (commercially available in South Africa) to a final con-entration of 2 g/100 ml, which compares well with the dose andupplementation time of the product used by Beltrán-Debón et al.2011). These concentrations are customary used for tea makingurposes. The mixture was allowed to cool at room temperature for0 min, filtered (Whatman no. 4) and dispensed into water bottles.

ntioxidant content and activity of the rooibos herbal teas andreen tea

The soluble solid content of the aqueous green tea and rooi-os extracts was determined gravimetrically (sixteen repetitions)

uring the course of the study after drying aliquots at 110 ◦C for2 h. The flavanol and flavonol contents of each extract were deter-ined spectrophotometrically at 640 nm and 360 nm, respectively

Treutter 1989; Mazza et al. 1999). The total polyphenol content

ne 18 (2011) 1220– 1228 1221

of the aqueous extracts was determined using the Folin Ciocal-teu method (Singleton and Rossi 1965), while the oxygen radicalabsorbance capacity (ORAC) was determined using a fluorometricmethod (Ou et al. 2001).

High-performance liquid chromatography analysis of the rooibosextracts

The tea samples were chromatographically separated using themethod described by Bramati et al. (2002) with modifications. AnAgilent Technologies 1200 Series HPLC system with a diode arraydetector and a 5 �m YMC-PackPro C18 (150 mm × 4.6 mm i.d.) col-umn was used for the separation. Acquisition was set at 287 and360 nm and the mobile phases consisted of water (A) containing300 �l/l trifluoroaceticacid and methanol (B) containing 300 �l/ltrifluoroaceticacid. The gradient elution started at 95% A changingto 75% A after 5 min and to 20% A after 25 min. The flow rate wasset at 0.8 ml/min, the injection volume was 20 �l and the columntemperature was set at 21 ◦C. Peaks were identified based on theretention time of the standards and confirmed by comparison ofthe wavelength scan spectra (set between 210 nm and 400 nm).

Animal experimental treatment

Male Wistar rats were used as experimental animals. Theyreceived humane care in accordance with the Principle of Labo-ratory Animal Care of the National Medical Research and the Guidefor the care and use of Laboratory animals of the National Academyof Sciences (National Institutes of Health Publications no. 80–23,revised 1978). The rats had free access to either the various aque-ous tea extracts or tap water for 7 weeks as their sole source ofdrinking fluid. The rats were fed standard rat chow (SRC) ad libi-tum, while the fluid intake was monitored on a daily basis. Therooibos and green tea extracts were freshly prepared every sec-ond day. The rats were housed in an experimental animal facilitykept at a constant temperature of 25 ◦C and exposed to a twelve-hour artificial day–night cycle. Ethical clearance for this study wasgranted by the Faculty of Health and Wellness Sciences’ ResearchEthics Committee of the Cape Peninsula University of Technology(Ref: CPUT/HAS-REC 2009/A001).

Male Wistar rats (120–150 g) were divided into four groupsfed standard rat chow (SRC) and supplemented with eithertap water (Control, C), unfermented/“green” rooibos (RU), fer-mented/traditional rooibos (RF) or green tea (GT) for a period ofseven weeks. At the end of the seven week feeding period, ani-mals were sacrificed. Hearts (n = 10 per group) were excised andmounted on the working heart perfusion apparatus and perfusedwith Krebs–Henseleit buffer. Mechanical function was documentedat 2 time points i.e. before ischaemia and 25 min after ischemia intoreperfusion. Aortic output recovery (AO) was the functional param-eter measured. Hearts were freeze clamped 10 min into reperfusionfor biochemical analysis of PKB/Akt, threonine and serine residues,caspase-3 and poly (ADP-ribose) polymerase (PARP), glutathione(GSH), glutathione disulfide (GSSG) and GSH/GSSG ratio as shownin Fig. 1.

Working heart perfusion protocol

After the feeding period, rats were anaesthetized using diethylether. Hearts were rapidly excised, then mounted on a workingheart perfusion apparatus and were perfused at a constant pressure

(75 mm Hg) using a Krebs–Henseleit buffer gassed with a mixtureof 5% carbon dioxide and 95% oxygen. After mounting, hearts weresubjected to 10 min stabilization, followed by 15 min of normoth-ermic global ischaemia and 25 min of reperfusion. Hearts were

1222 W.G. Pantsi et al. / Phytomedicine 18 (2011) 1220– 1228

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reeze-clamped 10 min into reperfusion and stored at −80 ◦C foriochemical analysis.

estern blot

Heart tissue was homogenized by adding homogenizationuffer and phenylmethanesulfonyl fluoride (PMSF) to the sam-le. For phosphoprotein determination sodium fluoride (NaF) andodium orthovanidate (Na3VO4) were added to the sample wherefter samples were sonicated 3 times for 10 s at a time and thenentrifuged at 15,000 rpm for 15 min. The protein concentrationsf the various homogenates were determined using the bicin-honinic acid method. Samples were diluted with Laemmli sampleuffer and boiled, after which 40 �g of protein was separatedy sodium dodecyl sulfate polyacrylamide gel electrophoresisSDS-PAGE). After electrophoresis the proteins were transferredo polyvinylidene fluoride (PVDF) membranes. Membranes wereoutinely stained with Ponceau in order to check for equal load-ng and sufficient transfer. Nonspecific binding was blocked byvernight incubation in 5% fat free milk in Tris-buffered saline with% tween (TBST). Membranes were then incubated with primaryntibodies that recognize total PKB/Akt, Ser 473, Thr 389, caspase-

and PARP. Membranes were subsequently washed and incubatedith a secondary antibody. After thorough washing with TBST,embranes were covered with a chromogenic substrate (Proteinetector BCIP/NBT Western Blotting Kit, Invitrogen). A photo of

he individual protein bands was captured and calculations werexpressed in pixels (arbitrary units).

lutathione analysis

Total glutathione (GSH and GSSG) was measured according to modified method of Asensi et al. (1999). Heart samples wereomogenized (1:25) in 15% (w/v) TCA containing 1 mM EDTA forSH determination and in 6% (v/v) PCA containing freshly prepared

mM M2VP and 1 mM EDTA for GSSG determination on ice. Afterentrifugation at 1400 rpm in 4 ◦C for 30 s, 50 �l of supernatant wasdded to glutathione reductase (1 U) and 75 �M DTNB. The reac-

ion was initiated by addition of 0.25 mM NADPH to a final volumef 200 �l. The change in absorbance was monitored at 410 nm for

min and levels were calculated using pure GSH and GSSG as stan-ards. The ratio of GSH/GSSG was also calculated using the results.

r functional and biochemistry measurements.

Statistical methods

Functional parameters are presented as percentage of the base-line values. Results are expressed as mean ± standard error of themean (SEM). Differences between two groups were determinedusing an unpaired Student’s t-test and to compare differences inmultiple groups a one-way ANOVA with a Bonferoni Multiple com-parison as a post hoc test was used. p < 0.05 was considered to bestatistically significant. Pearson correlation coefficients were calcu-lated when two sets of data were tested for a correlation betweenthe results of the different assays.

Results

Soluble solid content and daily intake of selected rooibos andgreen tea flavonoids

The soluble solids of GT were significantly (p < 0.05) higherwhen compared to the rooibos herbal teas (RF and RU). Ratsconsumed significantly (p < 0.05) more fermented/traditionalrooibos (RF) (69.00 ± 14.73 ml/day) and unfermented/“green”rooibos (RU) (55.10 ± 13.06 ml/day) when compared togreen tea (35.10 ± 4.33 ml/day). Rats that consumed the RF(67.70 ± 8.12 mg/day) ingested the lowest (p < 0.05) amountof total polyphenols compared to those consuming RU(74.62 ± 3.41 mg/day) and GT (95.58 ± 7.16 mg/day). Whenconsidering the flavanol and flavonol intake, the rats receiving GT(31.47 ± 0.89 mg/day) had a significantly higher flavanol intakecompared to rats consuming the RU (5.07 ± 0.14 mg/day) andRF (2.67 ± 0.56 mg/day). However, the flavonol intake of the ratsreceiving the GT (3.81 ± 0.56 mg/day) was significantly lower com-pared to RU (13.61 ± 1.07 mg/day) and RF (20.65 ± 3.41 mg/day).The daily ORAC (oxygen radical absorbance capacity) unit intakewere significantly higher (p < 0.05) in the GT groups whencompared to the RF and RU groups as shown in Table 1.

HPLC quantification of major flavonoids in the rooibos and greentea extracts

Only the major constituents of rooibos were quantified, withaspalathin being the major flavonoid in the unfermented/“green”rooibos extracts, and still one of the major flavonoids in the

W.G. Pantsi et al. / Phytomedicine 18 (2011) 1220– 1228 1223

Table 1Daily intake profile of the rooibos and green tea extracts.

Treatment C RF RU GT

Tea intake (ml/day) 74.50 ± 12.93 69.00 ± 14.73 a 55.10 ± 13.06 b 35.10 ± 4.33 c

Soluble solids (mg/ml) None 0.22 ± 0.08a 0.35 ± 0.03b 0.63 ± 0.05c

Polyphenol (mg/day) None 67.70 ± 8.12a 74.62 ± 3.41b 95.58 ± 7.16c

Flavanol (mg/day) None 2.67 ± 0.56a 5.07 ± 0.14b 31.47 ± 0.89c

Flavonol (mg/day) None 20.65 ± 3.41a 13.61 ± 1.07b 3.81 ± 0.56c

ORAC (�mole TE/day) None 210.96 ± 61.41a 379.11 ± 198.09b 950.16 ± 72.00c

Values in the rows represent the means ± SEM of 10 rats per group. Abbreviations: ORAC: oxygen radical absorbance capacity (ORAC), C: control, RF: fermented rooibos herbaltea, RU: unfermented rooibos herbal tea and GT: green tea and. Values followed by the different letters differ significantly (p < 0.05).

Fig. 2. HPLC chromatogram (287 nm and 360 nm) of flavonoids in unfermented rooibos: 1, aspalathin; 2, orientin; 3, isoorientin; 4, vitexin; 5, isovitexin; 6, rutin/hyperoside.

Fig. 3. HPLC chromatogram (287 nm and 360 nm) of flavonoids in fermented rooibos: 1, aspalathin; 2, orientin; 3, isoorientin; 4, vitexin; 5, isovitexin; 6, rutin/hyperoside;7, quercetin; 8, luteolin; 9, chrysoeriol.

Fig. 4. HPLC chromatogram (287 nm and 360 nm) of

flavonoids in green tea: 1, EGCG; 2, EC; 3, ECG.

1224 W.G. Pantsi et al. / Phytomedici

Fig. 5. Effect of dietary rooibos herbal tea supplementation on aortic output recov-ery (%) during reperfusion (n = 10 per group). Results were expressed as mean ± SEM(*p < 0.001 vs. C, $p < 0.001 vs. RU). AO, aortic output; C, control; RF, fermentedrooibos herbal tea; RU, unfermented rooibos herbal tea; GT, green tea.

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PARP cleavage in the RF (45.36 ± 2.67 pixels) and RU(47.82 ± 3.82 pixels) supplemented hearts is significantly (p < 0.05)

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ermented rooibos, together with orientin and isoorientin. Traceuantities of quercetin, luteolin and chrysoeriol are also present

n the fermented and unfermented rooibos extract (Table 2 andigs. 2 and 3). Epigallocatechin gallate (EGCG) was the majoravonoid in the green tea extract while EC and ECG were also

resent (Table 2 and Fig. 4).

ig. 6. (A) Effect of dietary rooibos herbal tea supplementation on total PKB/Akt in heartss mean ± SEM. (*p < 0.05 vs. C; #p < 0.05 vs. RF; &p < 0.05 vs. RU) C: control, RF: fermentedietary rooibos herbal tea supplementation on phosphorylated PKB/Akt on Thr 308 in hears mean ± SEM. (*p < 0.05 vs. C, #p < 0.05 vs. RF). C: control, RF: fermented rooibos herbaerbal tea supplementation on phosphorylated PKB/Akt on Ser 473 in hearts subjected to

*p < 0.05 vs. C, #p < 0.05 vs. RF). C: control, RF: fermented rooibos herbal tea, RU: unferme

ne 18 (2011) 1220– 1228

Function: aortic output (AO) recovery

AO recovery of hearts from the RU (61.58 ± 1.31%) and RF(61.06 ± 2.26%) supplemented groups was significantly (p < 0.05)increased when compared with the control (49.77 ± 1.83%) and GT(54.03 ± 1.50%) groups, respectively (Fig. 5).

Signaling pathway markers: protein kinase B (PKB/Akt), serine473(Ser 473) and threonine308 (Thr 308)

There was a significant (p < 0.05) increase in phosphoryla-tion of total PKB/Akt in GT (75.52 ± 3.07 pixels) supplementedhearts compared to all other groups, i.e. C (55.91 ± 4.01 pixels), RF(62.58 ± 2.097 pixels), and RU (55.51 ± 2.669 pixels) during reper-fusion (Fig. 6A). Fig. 6B shows that phosphorylation of Thr 308 wassignificantly increased in the hearts of GT (36.78 ± 3.045 pixels)supplemented group compared to the control (33.18 ± 2.045 pix-els) and RF (32.36 ± 1.47pixels) groups. Phosphorylation of Ser 473was significantly increased in the GT (43.31 ± 2.77 pixels) supple-mented hearts compared to the control (36.33 ± 4.01 pixels) and RF(37.32 ± 4.68 pixels) but not the RU (41.27 ± 4.92) group (Fig. 6C).

Apoptotic proteins: poly(ADP-ribose) polymerase (PARP) andcaspase-3

lower when compared to the control (54.37 ± 3.47 pixels) and GT

subjected to ischaemia and reperfusion. (n = 8 per group). Results were expressed rooibos herbal tea, RU: unfermented rooibos herbal tea, GT: green tea. (B) Effect ofts subjected to ischaemia and reperfusion (n = 8 per group). Results were expressedl tea, RU: unfermented rooibos herbal, GT: green tea. (C) Effect of dietary rooibosischaemia and reperfusion (n = 8 per group). Results were expressed as mean ± SEMnted rooibos herbal tea, GT: green tea.

W.G. Pantsi et al. / Phytomedici

Table 2Major rooibos and green tea flavonoids (mg/l) in the aqueous extracts quantified byHPLC.

Tea RF RU GT

Aspalathin 31.72 ± 3.78 361.84 ± 13.62 N.D.Orientin 24.43 ± 0.33 28.65 ± 4.63 N.D.Isoorientin 35.35 ± 0.49 44.93 ± 1.14 N.D.Vitexin 7.30 ± 0.18 7.03 ± 0.08 N.D.Isovitexin 7.99 ± 0.36 8.97 ± 0.23 N.D.Hyperoside/Rutin 13.38 ± 0.27 42.34 ± 0.28 N.D.Quercetin 2.16 ± 0.04 N.D. N.D.Luteolin 0.88 ± 0.01 N.D. N.D.Chrysoeriol 0.41 ± 0.01 N.D. N.D.Epigallocatechin gallate N.D. N.D. 156.11 ± 9.88Epicatechin N.D. N.D. 95.69 ± 5.29Epicatechin gallate N.D. N.D. 24.56 ± 3.01

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.D.: none detected.

54.39 ± 1.33 pixels) supplemented groups (Fig. 7A). Hearts of RF32.56 ± 0.78 pixels) and GT (30.87 ± 1.19 pixels) supplementedroups showed a significant (p < 0.05) decrease in caspase-3 activa-ion during reperfusion when compared with caspase-3 activationf hearts of the control group (43.80 ± 2.23 pixels). Caspase-3 acti-ation of hearts from the RU (53.41 ± 1.32 pixels) group showed

significant (p < 0.05) increase when compared to both RF and GTupplemented groups, with the control hearts (43.80 ± 2.23 pixels)

howing a significant decrease when compared to the RU supple-ented hearts (Fig. 7B).

ig. 7. (A) Effect of dietary rooibos herbal tea supplementation on PARP cleavagen hearts subjected to ischaemia and reperfusion. (n = 8 per group). Results werexpressed as mean ± SEM (*p < 0.05 vs. C, #p < 0.05 vs. GT). C: control, RF: fermentedooibos herbal tea, RU: unfermented rooibos herbal tea, GT: green tea. (B) Effect ofietary rooibos herbal tea supplementation on caspase-3 cleavage in hearts sub-

ected to ischaemia and reperfusion (n = 8 per group). Results were expressed asean ± SEM (*p < 0.05 vs. C). C: control, RF: fermented rooibos herbal tea, RU: unfer-ented rooibos herbal tea, GT: green tea.

ne 18 (2011) 1220– 1228 1225

Reduced glutathione (GSH) and oxidized glutathione (GSSG) levels

The tGSH level in the GT (3.22 ± 1.08 �M/g) supplementedgroup was significantly (p < 0.05) lower when compared tothe control (7.45 ± 0.74 �M/g), RF (6.82 ± 2.05 �M/g) and RU(8.74 ± 1.09 �M/g) groups. Furthermore, the tGSH level in theRU supplemented group was significantly (p < 0.05) higher thanin the RF supplemented group (Fig. 8A). The GSSG level in theRF (0.48 ± 0.37 �M/g) and GT (0.62 ± 0.17 �M/g) supplementedgroups was significantly (p < 0.05) higher when compared tothe control (0.13 ± 0.08 �M/g) and RU (0.373 ± 0.06 �M/g) groups(Fig. 8B). The GSH/GSSG ratio in the hearts of the GT (10.58 ± 7.48)supplemented group was significantly (p < 0.05) lower when com-pared to that of the RF (37.78 ± 28.63), RU (33.20 ± 4.13) and control(45.50 ± 14.96) groups (Fig. 8C).

Discussion

Rooibos is a rich source of unique antioxidants due to itsflavonoid content (Joubert et al. 2005). Flavonoids are known toprotect against myocardial ischemia/reperfusion injury by theirmultifaceted properties, such as anti-inflammatory, antioxidant,vasodilatory, and antiplatelet aggregation (Akhlaghi and Band2009). The flavonoid constituents of unfermented/“green” rooibos(RU) and fermented/“traditional” rooibos (RF) differ from those ofgreen tea (GT). Although, the cardioprotective effect of green teawas demonstrated in other animal studies (Babu et al. 2006; Pasiniet al. 2009). The data obtained in this study show, for the first time,that both RF and RU supplemented hearts are able to improve aorticoutput (AO) recovery when compared to the control groups withGT supplemented hearts not showing any improved AO recoverywhen compared to the control. The antioxidant quantitative datafrom the present study show that the flavonol content of RU and RFare higher when compared to that of GT, with flavanol content of GThigher than that of RU and RF. A good correlation (r = 0.9276) wasalso shown between the aortic output recovery and flavonol con-tent but not the flavanol content of the extracts. It could thereforebe concluded that rats consuming the RF and RU showed improvedAO recovery associated with the higher intake of flavonols. Thisargues that in our model, rooibos flavonols are more effective asprotectors against ischaemia/reperfusion injury compared to greentea flavanols. Animal studies that have investigated the cardio-protective effects of natural or synthetic flavonoids have focusedmainly on the acute pharmacological activity of these compounds.For example, in vivo studies using animal models have reported onacute cardioprotection obtained from intravenous injections of nat-ural or synthetic flavonoids (Ji et al. 2004; Wang et al. 2004). Morerecently, results from human studies provide evidence that rooi-bos can offer protection against oxidative stress conditions suchas cardiovascular disease (Villano et al. 2010; Marnewick et al.2011). To our knowledge, no investigation has been done regardingthe effect of rooibos flavonols on the isolated perfused heart afteran episode of ischaemia/reperfusion injury. The increased phos-phorylation of protein kinase B (PKB/Akt) on serine 473 (Ser 473)and threonine 308 (Thr 308) by GT, could be attributed to the factthat GT has more flavanols and a higher polyphenol content com-pared to RU and RF. Correlation analysis also showed a negativecorrelation (r = −0.814) between the flavonol content of the rooi-bos extracts and total PKB/Akt, This finding may suggest that theflavonol content of RF and RU had little effect on phosphorylationof PKB/Akt. Evidence is accumulating suggesting that the cellular

effects of flavonoids may be mediated by their interactions withthe PKB/Akt signaling pathway cascade (Schroeter et al. 2002). Inaddition, EGCG, the main catechin found in GT, could have an effecton phosphorylation of PKB/Akt.

1226 W.G. Pantsi et al. / Phytomedicine 18 (2011) 1220– 1228

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ig. 8. Effect of dietary rooibos herbal tea supplementation on GSH analysis in heaean ± SEM. A: (*p < 0.05 vs. C, #p < 0.05 vs. RF, &p < 0.05 vs. RU). C: control, RF: ferm

s. C). C: (*p < 0.05 vs. C, $p < 0.05 vs. RF).

Apoptosis has been consistently observed in cardiomyocytesfter reperfusion and may represent a mechanism by which car-iac cells are damaged (Abe et al. 2000). Our results show thatoth rooibos extract (RF and RU) supplemented rat hearts sig-ificantly decreased PARP cleavage compared to the control andreen tea supplemented hearts, showing a negative correlationr = −0.9979) between the flavonol content of the aqueous extractsnd the decreased PARP cleavage. This correlation could not behown for the flavanol content of the extracts. This finding stronglyuggests that the protection offered by RU and RF may be as aesult of inhibition of apoptosis. It would appear under the currentonditions, that the herbal tea extracts with the highest flavonolontent are likely to attenuate PARP cleavage. However, our resultshow that GT, with the highest polyphenol content than bothU and RF, had no effect on PARP cleavage. Rat hearts supple-ented with RU and RF showed a significant decrease in cleaved

ARP after 10 min into reperfusion, compared to other groups. Thisrovides circumstantial evidence to show that rooibos flavonolsre highly effective in protecting against ischaemia/reperfusionnjury. These results are also supported by the improved AO recov-ry at 25 min into reperfusion in the RU and RF supplementedroups. This finding may therefore argue that decreased PARPleavage may be a possible mechanism of protection offered by RFnd RU.

Caspase-3 is one of the key executioners of apoptosis, capa-le of cleaving or degrading many key proteins including nuclear

amins, fodrin as well as PARP (Lawen 2003). Recent studies havehown that quercetin, a flavonol, can prevent apoptosis by alter-ng the expression of Bax, Bcl-2 and caspase-3 (Chao et al. 2009).he present study showed that caspase-3 cleavage in the RF groupas significantly decreased when compared to the control group.

his was also associated with a decrease on PARP cleavage, stronglyrguing for an anti-apoptotic role for RF. Our results are also ingreement with those obtained for quercetin, which suppresses

he expression of cleaved caspase-3 in the liver (Liu et al. 2010).urthermore, our results suggested that rooibos flavonoids couldlleviate ischaemia/reperfusion injury by suppressing apoptosis. Inddition, the higher flavonol content of RF may contribute to the

bjected to ischaemia and reperfusion (n = 8 per group). Results were expressed as rooibos herbal tea, RU: unfermented rooibos herbal tea, GT: green tea. B: (*p < 0.05

attenuation of PARP cleavage. However, in the RU group, caspase-3cleavage was significantly increased. Since PARP cleavage in the RUgroup was decreased, this result needs to be further investigated.It is known that caspase-3 has a direct effect on PARP cleavageand that these two proteins should change in the same direction.Although this is not the case in the current study, it may be arguedthat one or more of the many components of RU could have influ-enced cleavage of caspase-3 differently, but further investigation isneeded.

While beneficial effects of dietary rooibos flavonoids wereobserved ex vivo in isolated perfused rat hearts, it is unlikely that theprotection might be due to a direct antioxidant effect of the circu-lating rooibos flavonoids or their respective metabolites. It has beensuggested that flavonoids might affect endogenous antioxidantdefenses of cells by modulating the glutathione and glutathionerelated enzyme systems (Masella et al. 2005). In the present study,the tGSH level was significantly higher in the RU and RF groupcompared to the GT. This may suggest that, in the present study,flavonols have an effect on the tGSH levels. Despite the higherpolyphenol, flavanol and ORAC values in the GT fed rats, tGSH levelsin this group were lower when compared to RU and RF supple-mented groups. In the GT supplemented group, the GSSG levelswere increased in the heart, resulting in a decrease of the GSH/GSSGratio. It is known that the GSH/GSSG ratio decreases under stressconditions due to increased GSSG levels or as a result of decreasedGSH levels (Dickson and Forman 2002). Changes in the GSH andGSSG levels resulted in a significantly reduced GSH/GSSG ratio inGT fed rats compared to the control, RF and RU fed rats. Both rooi-bos extracts caused a significant recovery of this ratio (similar tothat of the negative control) when compared to the rats consumingthe green tea. The significant decrease in GSH/GSSG ratio could beassociated with the poor AO recovery in GT fed rats. Green tea inthe current study did not improve aortic output recovery despitethe higher flavanol, polyphenol content and ORAC. Even though

not significantly, RU and RF showed a higher GSH/GSSG ratio whencompared to GT and comparable to that of the control group. Thiswas also associated with improved AO recovery in the RU and RFfed hearts.

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Most significant of the current findings, is that it confirms resultsy Beltrán-Debón et al. (2011) in a hyperlipidemic mouse model. Inhe Beltron-Debon model it was concluded that cellular signalingathways could be involved in the beneficial effects of rooibos. Also

t is interesting to note that the daily dosage and the feeding periodere similar if you closely compare the two models used. Beltrán-ebón et al. (2011) used a daily dose of 10 g/l in a mouse model

or 14 weeks and in the current study 2 g/100 ml/day for 7 weeks.hese findings may suggest that rooibos extract can be used as aherapeutic substance in oxidative stress related diseases.

In conclusion, rooibos has been shown to favorably modu-ate oxidative stress, the plasma lipid profile and antioxidanttatus (Villano et al. 2010; Marnewick et al. 2011), metabolicomplications (Beltrán-Debón et al. 2011) as well as inhibithe activity of angiotensin-converting enzyme with no effectn nitric oxide in humans (Persson et al. 2010). Results fromhe present study showed rooibos to protect against myocardialschaemia/reperfusion injury in vivo and, taking into account pre-ious results, it is suggested that the unique combination of rooibosavonoid compounds have beneficial effects on vascular function.he protective effect could partially be due to the antioxidantffects of the rooibos polyphenols, in particular flavonols, and tar-eting specific cellular pathways such as the apoptotic pathway.owever, further studies are needed to determine the vascu-

ar effects of specific rooibos compounds and/or their respectiveetabolites as well as to confirm that rooibos could be a potential

herapeutic substance.

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